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Several procedures to isolate DNA from difficult sources have previously been described, but they are often expensive, time consuming, and have limited applications. In this paper we describe a simple and versatile protocol to isolate nucleic acids from different plant tissues, using a silica-based extraction method. This extraction process efficiently purifies DNA from several plant cells. The obtained DNA has successfully been applied in polymerase chain reaction (PCR) assays for OGM purposes. The protocol is compatible with at least two automated liquid handling systems making it suitable for large-scale screening applications.

Congenital Hyperinsulinism of Infancy (CHI) is a genetically heterogeneous disorder characterized by profound hypoglycemia related to inappropriate insulin secretion. Two histopathologically and genetically distinct groups are recognized among patients with CHI due to ATP-sensitive potassium channel (KATP) defects: a diffuse type (Di-CHI), which involves the whole pancreas, and a focal form (Fo-CHI), which shows adenomatous islet-cell hyperplasia of a particular area within the normal pancreas. The beta-cell KATP channel consists of two essential subunits: Kir6.2 encoded by the KCNJ11 gene which is the pore-forming unit and belongs to the inwardly rectifying potassium channel family, and SUR1 (sulfonylurea receptor 1) encoded by the ABCC8 gene, which belongs to the ATP-binding cassette (ABC) transporter family. The KATP channel is an octameric complex of four Kir6.2 and four SUR1 subunits. More than one hundred mutations have been found in KATP channel genes ABCC8 and KCNJ11 , but to date only twenty mutations have been identified in KCNJ11 , most of them are missense mutations and only one is a single base deletion. The Fo-CHI has been demonstrated to arise in individuals who have a germline mutation in the paternal allele of ABCC8 or KCNJ11 in addition to a somatic loss of the maternally derived chromosome region 11p15 in adenomatous pancreatic β-cells, while Di-CHI predominantly arises from the autosomal recessive inheritance of KATP channel gene mutations. Here we describe the molecular findings in nine children who presented, in the neonatal period, with signs and symptoms of hypoglycemia and diagnosed affected by CHI according to international diagnostic criteria. Direct sequencing of the complete coding exon and promoter region of KCNJ11 gene showed, in two Italian patients, two new heterozygous mutations which result in the appearance of premature translation termination codons resulting in the premature end of Kir6.2. Interestingly most of the CHI mutations detected in other population studies are situated in the ABCC8 gene.

Since the cloning in 1997 of SEL1L, the human ortholog of the sel-1 gene of C. elegans, most studies have focused on its role in cancer progression and have provided significant evidences to link its increased expression to a decrease in tumor aggressiveness. SEL1L resides on a “Genome Desert area” on chromosome 14q24.3-31 and is highly conserved in evolution. The function of the SEL1L encoded protein is still very elusive although, several evidences from lower organisms indicate that it plays a major role in protein degradation using the ubiquitin-proteosome system. SEL1L has a very complex structure made up of modules: genomically it consists of 21 exons featuring several alternative transcripts encoding for putative protein isoforms. This structural complexity ensures protein flexibility and specificity, indeed the protein was found in different sub-cellular compartments and may turn on a particular transcript in response to specific stimuli. The overall architecture of SEL1L guarantees an exquisite regulation in the expression of the gene. © 2005 Wiley-Liss, Inc.

SEL1L, a human gene located on chromosome 14q24.3–q31, is highly expressed in adult pancreas. It is proximal to D14S67 (IDDM11) a proposed type I diabetes susceptibility locus. Considering the organ specific expression of SEL1L, a fundamental role of SEL1L in pancreatic growth can be hypothesized. While screening for mutations in young diabetic patients, in children affected by persistent hyperinsulinemic hypoglycemia of infancy (PHHI), in patients with non-functional endocrine tumours and in over 100 control subjects, we identified a novel polymorphism (D162G) residing on the fourth exon of the gene. This exon encodes for the fibronectin type II domain and the nucleotide change involves a highly conserved amino acid. The D162G polymorphism induces a major change in the amino acid composition producing a possible disruptive role in collagen binding.